Equine encephalosis is an arthropod-borne, noncontagious, febrile
disease of horses. It was first described >100 years ago by A.
Theiler (1) under the name equine ephemeral fever. The disease is caused
by Equine encephalosis virus (EEV; genus Orbivirus: subfamily
Sedoreovirinae: family Reoviridae) (2,3), which is transmitted by
Culicoides spp. biting midges (4). Before 2008, EEV had been isolated
only in South Africa, where 7 antigenically distinct serotypes, EEV-1-7,
have been identified and characterized (3).

Orbiviruses encode at least 7 structural and 4 nonstructural (NS)
proteins from 10 linear dsRNA genome segments (5). The smallest genome
segment, segment 10 (Seg-10), encodes NS3, which mediates the release of
virus particles from infected cells, and NS3A. The second largest of the
EEV genome segments, Seg-2, encodes virus protein (VP) 2, the larger of
the 2 outer-capsid proteins. By analogy with bluetongue virus (BTV), the
Orbivirus type species, the virus serotype is determined by the
specificity of interactions between VP2 and neutralizing antibodies
generated during infection of the mammalian host. Consequently, VP2 and
Seg-2 show sequence variations that correlate with serotype and, thus,
can be used to determine the virus serotype (6).

From October 2008 through January 2009, a febrile horse disease
that was diagnosed as equine encephalosis was observed in dozens of
stables across Israel (7). The recent emergence of novel orbivirus
strains (including BTV and epizootic hemorrhagic disease virus) in
Europe, North America, Asia, and Australia (8) is of major concern to
the worldwide livestock industry. Furthermore, the similarity of EEV to
African horse sickness virus, one of the most devastating pathogens of
equids, warranted further investigation of the outbreaks and molecular
characterization of the virus. The molecular and sequence analyses
reported here confirm the existence of EEV in Israel and identify the
virus and its serotype, as well as its phylogenetic roots.

Total RNA was extracted from the fifth and sixth passages of all 3
samples by using the QIAamp Viral RNA Mini Kit (QIAGEN, Valencia, CA,
USA) to obtain sufficient viral load for the subsequent analyses and
replications. RNA was reverse transcribed into cDNA by using the Verso
cDNA Kit (Thermo Fisher Scientific, Epsom, UK). PCR amplification of the
gene encoding NS3 (Seg-10) was performed on the 3 isolates by using
GoTaq Green Master Mix (Promega, Madison, WI, USA) with the following
primers: 5'-[sup.1]GTT AAG TTT CTG CGC CAT GT[sup.23]-3',
5'-[sup.741]GTA ACA CGT TTC CGC CAC G[sup.760]-3'. Thermal
cycling conditions for the PCR were as previously described (9); the
primer annealing temperature was modified to 53.5[degrees]C. PCR
products were purified by using a cDNA purification kit (ExoSAP-IT; USB,
Cleveland, OH, USA), and sequencing was conducted by BigDye terminator
cycle sequencing chemistry (Applied Biosystems, Foster City, CA, USA) in
an ABI 3700 DNA Analyzer (Applied Biosystems) by using ABI data
collection and sequence analysis software. Further analysis of the NS3
sequence was performed with Sequencer software, version 4.8 (Gene Codes
Corp., Ann Arbor, MI, USA). Sequences were deposited in GenBank under
accession nos. HQ441245 for H5, HQ441246 for H3, and HQ441247 for H8.
The NS3 genes (Seg-10) were compared with those of different EEVs (9)
and other related orbiviruses (Table 2). Phylogenic trees were generated
by using the neighbor-joining and maximum-likelihood methods (Phylip
Inference Package version 3.68, Seqboot Program; J. Felsenstein,
University of Washington, Seattle, WA, USA) to create 100 datasets
(bootstrapping) and the DNA Maximum Likelihood Program version 3.5
(http:// cmgm.stanford.edu/phylip/dnaml.html) to construct the trees.
Finally, the Consense program version 3.5c (http://
cmgm.stanford.edu/phylip/consense.html) was used to create a final
consensus tree for our dataset. Broadhaven virus, a tick-borne
orbivirus, was used as the outgroup in the phylogram for the gene
encoding NS3.

[FIGURE 1 OMITTED]

The phylogenetic analyses of EEV Seg-10 grouped the Israeli
isolates with other EEV isolates but as a distinct group with no close
relation to African horse sickness virus, BTV, or epizootic hemorrhagic
disease virus. Within the EEV group, 3 discrete clusters (A, B, C) were
recognized; the Israeli isolates formed one of these clusters (C; Figure
2). The Israeli isolates have 85%-86% nt identity to cluster A and
75%-76% nt identity to cluster B.

In addition, full-length cDNA copies of individual EEV (from H3 and
H8) genome segments were synthesized and amplified by reverse
transcription PCR by using the anchor spacer-ligation method as
described (10,11). Partial sequences (for the upstream 450 bp) of Seg-2
from the different Israeli isolates were identical, showing 92.3% nt and
95.7% aa sequence identity with Seg-2 and VP2 of the Kaalplaas isolate,
the reference isolate of EEV-3 (GenBank accession numbers are listed in
Table 2). Previous phylogenetic comparisons of Seg-2/VP2 from different
BTV types showed a maximum of 71% nt and 78% aa acid identity between
serotypes (6), indicating that the isolates from Israel also belong to
EEV type 3.

Conclusions

Equine encephalosis virus has long been enzootic to southern
Africa, but it has not been isolated in other parts of the world. We
report the characterization of an EEV strain isolated outside of Africa.
Phylogenetic analysis of Seg-2 showed 92% sequence identity to EEV-3
(Kaalplaas).

Analysis of Seg-10 (the gene encoding NS3) of different orbiviruses
showed 2 clusters of South African EEV strains (A and B), in agreement
with previously published studies (9). These 2 clusters appear to
correlate with the geographic origins of the viruses in South Africa,
independent of their isolation date. It has been suggested that the 2
EEV Seg-10 clusters in South Africa are related to the distribution of
their Culicoides spp. midge vectors, C. imicola (senso stricto) and C.
bolitinos. The former is the most abundant Culicoides spp. midge in
Israel (12). However, the EEV isolates from Israel group as a distinct
cluster (C) with similar distances to the 2 South African clusters,
raising questions concerning the geographic origin of this virus. A
similar finding has been observed in African horse sickness virus
Seg-10, which also forms into 3 distinct groups (13).

The question of how and when the virus was initially introduced to
Israel remains unanswered. Because the clinical manifestations of equine
encephalosis are usually mild, it is often overlooked and
underdiagnosed. EEV could have been introduced to Israel before the
virus was first isolated in 2009. Alternatively, the virus might have
been introduced into neighboring countries and transmitted into Israel
by infected vectors carried by winds, as described for other orbiviruses
(14,15). The fact that the Israeli strain of EEV-3 grouped in a
different cluster than the 2 South African strains, supports the idea
that it has evolved in this region for a sufficient time to accumulate
these changes and most likely was not recently introduced into Israel
from South Africa.

[FIGURE 2 OMITTED]

Acknowledgment

We thank Irit Orr for helping with the phylogenetic analysis.

Test development and analyses at Institute for Animal Health
Pirbright were supported by Department for Environment, Food, and Rural
Affairs, Biotechnology and Biological Sciences Research Council, and by
European Union contracts OrbiVac-245266, WildTech-222633-2, and
OrbiNet-K1303206.

Dr Aharonson-Raz is a veterinarian and a PhD candidate at the Koret
School of Veterinary Medicine, Israel. Her primary research interest is
epidemiology of arboviruses and infectious diseases of horses.